HIV presents a major obstacle to human health, particularly in developing nations. Large strides have been made in understanding the basic molecular biology of HIV, which have led to advances in the development and success of antiretroviral therapies. Yet despite this knowledge, there are still many aspects of HIV biology which we do not understand. If we are to ever truly cure individuals of HIV, we must first fully understand the molecular mechanisms of viral replication to develop novel therapies that take advantage of essential steps in this lifecycle. One such aspect of HIV biology that has remained a mystery despite decades of research is the accessory gene Vpr. Vpr is evolutionarily conserved and important for pathogenesis in vivo, yet no clear role for Vpr in viral replication has been defined. An emerging property of Vpr-associated phenotypes is engagement of the DNA damage response (DDR). The DDR is a signaling cascade that is vital to ensuring the fidelity of the host genome in the presence of genotoxic stress. Growing evidence has emphasized the importance of both activation and repression of the host DDR by diverse DNA and RNA viruses. However, precisely how and why Vpr engages the DDR is unclear. We have recently begun to bridge this gap in knowledge by identifying that Vpr both activates and represses the DDR at multiple steps. Specifically, we have found that Vpr represses the ability of the cell to repair double- strand DNA breaks via homologous recombination (HR) and non-homologous end joining (NHEJ). Based on our preliminary data, we hypothesize that repression of double-strand DNA break repair is central to the primary function of Vpr. Moreover, we propose that the inability of Vpr-expressing cells to repair damaged DNA represents a tractable means to selectively deplete HIV+ cells via synthetic lethality with genotoxic agents that induce low levels of additional DNA damage. We will take a combined molecular, proteomic, and evolutionary approach to directly test our hypotheses. Success of our proposed research will define the primary role of Vpr, it will elucidate how the DDR regulates HIV replication, and it will provide a novel means to treat HIV+ individuals and clear infected cells.